We are developing a novel class of non-surfactant microbicidal agents, the polybiguanides (PBG) that perturb membrane structure, redistribute membrane-associated proteins (including HIV-1 co-receptors and alter virus/host cell interactions. In Project III, we propose to assess the anti-HIV-1 activity of polyethylene, hexamethylene biguanide (PEHMB)-based microbicides and combination microbicides synthesized, purified, and characterized in Project I. Lead compounds for testing in Project III will be those previously assessed as non-toxic in Project II. The HYPOTHESIS is that computational chemistry approaches combined with rationale drug design will result in single or combination formulations of PEHMB-based microbicides that exhibit low toxicity with a high degree of anti-HIV-1 activity. To this end, we are combining PEHMB-compounds with non-toxic, highly active sulfated dendrimers in order to produce microbicide formulations that interdict HIV-1 infection by multiple mechanisms of action. The SPECIFIC AIMS of Project III are to (1) quantitate the anti-HIV-1 activity of non-formulated and formulated PEHMB-based and PEHMB-dendrimer combination based compounds (synthesized in Project I) with respect to inactivation of cell-free virus (CFI), inactivation of cell-associated virus (CAI) within infected cells of lymphocytic or monocytic origin and inhibition of viral binding and entry (VBI); 2.) determine the efficacy of PEHMB-based and PEHMB-dendrimer combination based compounds (synthesized in Project I) with respect to inhibiting HIV-1 infection of human vaginal cellular targets (lymphocytes, monocytes, dendritic cells, and epithelial cells) cultured in vitro utilizing both cell-free and cell-associated viral inoculums; 3.) Utilize an in vitro organ culture model of vaginal epithelium to examine the penetration of infectious HIV-1 through artificial vaginal formulations containing select PEHMB-based and PEHMB-dendrimer combination based compounds (identified in Aims 1 and 2) to an underlying permissive target cell monolayer and 4.) measure the ability of select PEHMB-based and PEHMB-dendrimer combination based compounds (identified in Aims 1 and 2) to block infections by cell-free and cell-associated HIV-1 in two models of virus infection: a mouse vaginal hu-PBL-NOD-SCID model and a human vaginal epithelial tissue xenograft model in NOD-SCID mice. The studies _erformed in Project III will be pursued in consultation with investigators in Project I and Project II, who will be involved in a detailed assessment of the cellular sensitivity of PEHMB-based formulations utilizing both in vitro and in vivo model systems. Results obtained in Projects II and III will be utilized to derive in vitro therapeutic indices for the selected PEHMB-based compounds that will be constructed in Project I. These results will be useful in selecting the most promising compounds to examine in the HIV-l-infected human vaginal xenograft model and for advancement to clinical trials in humans.